The invention relates to a device for preparing specimens for a cryo-electron microscope, comprising an environmental chamber, a holder for a sample or a carrier, and at least one blotting element to which a medium for absorbing liquid is or can be attached, both the holder and the blotting element(s) being disposed in the environmental chamber, and a cooling medium, preferably a container for a fluid, for cooling down the sample.
Besides the scanning electron microscope (SEM), wherein a scanning electron beam is used mainly for studying surfaces, various types of transmission electron microscopes (TEM) are used. With several of such microscopes, samples, for example a biopt or a cell suspension, are prepared by fixing and de-watering. After embedding a sample in a resin, an ultra-thin section (for example of 50 nm) can be cut, which is subsequently dyed and exposed to an electron beam.
In cryo-electron microscopy it is generally preferred to employ a so-called grid. For instance, a grid comprising hexagonal openings having a diameter of a few dozen microns is immersed in a suspension of particles to be examined, such as protein molecules or cells. Following that, the excess suspension is removed by “blotting”, i.e. by pressing blotting paper or another medium that readily absorbs liquid against the grid on one side or on both sides thereof. As a result of blotting, the thickness of the film that is present in the openings of the grid is reduced, for example from 3-4 μm to 100 nm. In a film of such minimal thickness, an area which is even thinner develops in the centre of the film under the influence of the London/van der Waals attraction of the air/fluid boundary layers, which area slowly expands in outward direction, that is, in the direction of the edge of the respective opening in the grid. During this process, which is also referred to as draining, the suspended particles are trapped between the fluid surfaces. As a next step, the sample is cooled down very quickly, so that the fluid becomes vitreous. The specimen thus prepared can be examined in a cryo-electron microscope at a low temperature (for example minus 170° C.) and be photographed without any additional operations being required.
A device for preparing specimens for a cryo-electron microscope is known, for example, from S. Trachtenberg, “A Fast-Freezing Device with a Retractable Environmental Chamber, Suitable for Kinetic Cryo-Electron Microscopy Studies”, Journal of Structural Biology 123, 45-55 (1998). Said publication describes a device which comprises an environmental chamber having transparent polycarbonate walls. Disposed under said chamber is a bath of liquid ethane. The temperature and the (high) air humidity in the chamber are controlled by means of heating elements and an ultrasonic air humidifier. After a grid has been provided with a sample, blotting takes place either manually or automatically by means of an independent unit. Then a valve present in the bottom of the chamber is opened and the grid falls into the ethane at high speed, so that the sample cools down very quickly, i.e. vitrifies. Following that, the chamber is moved up and the specimen is ready.
A similar device is known from a PhD thesis by J. Bednar, “Cryo-Electron Microscopy of DNA and Chromatin”, Lausanne 1995. This thesis describes (especially in Chapter 7 in conjunction with FIG. 7.1) a “plunger . . . (which) consists of stand., humid chamber, tweezers holder, and blotting pad as major parts”. The blotting pad is connected with a driving magnet via a photographic wire remote shutter.
Such arrangements and method suit the perception that the specimen is sensitive to changes in the temperature and air humidity levels until the moment of freezing. It has become apparent, however, that the existing equipment is insufficiently capable of preparing large numbers of specimens in a reproducible manner.